This invention is in the field of viral constructs for gene delivery. More specifically, the invention is in the field of recombinant DNA constructs for use in the production of adeno-associated virus (AAV) vectors for gene delivery.
Vectors based on adeno-associated virus (AAV) are believed to have utility for gene therapy but a significant obstacle has been the difficulty in generating such vectors in amounts that would be clinically useful for human gene therapy applications. This is a particular problem for in vivo applications such as direct delivery to the lung. Another important goal in the gene therapy context, discussed in more detail herein, is the production of vector preparations that are essentially free of replication-competent virions. The following description briefly summarizes studies involving adeno-associated virus and AAV vectors, and then describes a number of novel improvements according to the present invention that are useful for efficiently generating high titer recombinant AAV vector (rAAV) preparations suitable for use in gene therapy.
Adeno-associated virus is a defective parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. General reviews of AAV may be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. I, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). Examples of co-infecting viruses that provide helper functions for AAV growth and replication are adenoviruses, herpesviruses and, in some cases, poxviruses such as vaccinia. The nature of the helper function is not entirely known but it appears that the helper virus indirectly renders the cell permissive for AAV replication. This belief is supported by the observation that AAV replication may occur at low efficiency in the absence of helper virus co-infection if the cells are treated with agents that are either genotoxic or that disrupt the cell cycle.
Although AAV may replicate to a limited extent in the absence of helper virus, under such conditions as noted above, more generally infection of cells with AAV in the absence of helper functions results in the proviral AAV genome integrating into the host cell genome. Unlike other viruses, such as many retroviruses, it appears that AAV generally integrates into a unique position in the human genome. Thus, it has been reported that, in human cells, AAV integrates into a unique position (referred to as an xe2x80x9cAAV integration sitexe2x80x9d) which is located on chromosome 19. See, e.g., Weitzman et al. (1994) Proc. Nat""l. Acad. Sci. USA 91: 5808-5812. If host cells having an integrated AAV are subsequently superinfected with a helper virus such as adenovirus, the integrated AAV genome can be rescued and replicated to yield a burst of infectious progeny AAV particles. A sequence at the AAV integration site, referred to as xe2x80x9cP1,xe2x80x9d shares homology with the AAV inverted terminal repeat (ITR) sequence, exhibits activity in a cell-free replication system, and is believed to be involved in both the integration and rescue of AAV. See, e.g., Weitzman et al., id., Kotin et al. (1992) EMBO J. 11:5071-5078, and Urcelay et al., J. Virol. 69: 2038-2046. The fact that integration of AAV appears to be efficient and site-specific makes AAV a useful vector for introducing genes into cells for uses such as human gene therapy.
AAV has a very broad host range without any obvious species or tissue specificity and can replicate in virtually any cell line of human, simian or rodent origin provided that an appropriate helper is present. AAV is also relatively ubiquitous and has been isolated from a wide variety of animal species including most mammalian and several avian species.
AAV is not associated with the cause of any disease. Nor is AAV a transforming or oncogenic virus, and integration of AAV into the genetic material of human cells generally does not cause significant alteration of the growth properties or morphological characteristics of the host cells. These properties of AAV also recommend it as a potentially useful human gene therapy vector because most of the other viral systems proposed for this application, such as retroviruses, adenoviruses, herpesviruses, or poxviruses, are disease-causing.
Although various serotypes of AAV are known to exist, they are all closely related functionally, structurally, and at the genetic level (see, e.g., Blacklow, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison (ed.); and Rose, 1974, Comprehensive Virology 3: 1-61). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to inverted terminal repeats (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Thus, although the AAV2 serotype was used in various illustrations of the present invention that are set forth in the Examples, general reference to AAV herein encompasses all AAV serotypes, and it is fully expected that the methods and compositions disclosed herein will be applicable to all AAV serotypes.
AAV particles are comprised of a proteinaceous capsid having three capsid proteins, VP1, VP2 and VP3, which enclose a DNA genome. The AAV2 DNA genome, for example, is a linear single-stranded DNA molecule having a molecular weight of about 1.5xc3x97106 daltons and a length of about 5 kb. Individual particles package only one DNA molecule strand, but this may be either the xe2x80x9cplusxe2x80x9d or xe2x80x9cminusxe2x80x9d strand. Particles containing either strand are infectious and replication occurs by conversion of the parental infecting single strand to a duplex form and subsequent amplification of a large pool of duplex molecules from which progeny single strands are displaced and packaged into capsids. Duplex or single-strand copies of AAV genomes can be inserted into bacterial plasmids or phagemids and transfected into adenovirus-infected cells; these techniques have facilitated the study of AAV genetics and the development of AAV vectors.
The AAV genome, which encodes proteins mediating replication and encapsidation of the viral DNA, is generally flanked by two copies of inverted terminal repeats (ITRs). In the case of AAV2, for example, the ITRs are each 145 nucleotides in length, flanking a unique sequence region of about 4470 nucleotides that contains two main open reading frames for the rep and cap genes (Srivastiva et al., 1983, J. Virol., 45:555-564; Hermonat et al., J. Virol. 51:329-339; Tratschin et al., 1984a, J. Virol., 51:611-619). The AAV2 unique region contains three transcription promoters p5, p19, and p40 (Laughlin et al., 1979, Proc. Natl. Acad. Sci. USA, 76:5567-5571) that are used to express the rep and cap genes. The ITR sequences are required in cis and are sufficient to provide a functional origin of replication (ori), signals required for integration into the cell genome, and efficient excision and rescue from host cell chromosomes or recombinant plasmids. It has also been shown that the ITR can function directly as a transcription promoter in an AAV vector. See Flotte et al., 1993, supra; and Carter et al., U.S. Pat. No. 5,587,308.
The rep and cap gene products are required in trans to provide functions for replication and encapsidation of viral genome, respectively. Again, using AAV2 for purposes of illustration, the rep gene is expressed from two promoters, p5 and p19, and produces four proteins. Transcription from p5 yields an unspliced 4.2 kb mRNA encoding a first Rep protein (Rep78), and a spliced 3.9 kb mRNA encoding a second Rep protein (Rep68). Transcription from p19 yields an unspliced mRNA encoding a third Rep protein (Rep52), and a spliced 3.3 kb mRNA encoding a fourth Rep protein (Rep40). Thus, the four Rep proteins all comprise a common internal region sequence but differ in their amino and carboxyl terminal regions. Only the large Rep proteins (i.e. Rep78 and Rep68) are required for AAV duplex DNA replication, but the small Rep proteins (i.e. Rep52 and Rep40) appear to be needed for progeny, single-strand DNA accumulation (Chejanovsky and Carter, 1989, Virology 173:120-128). Rep68 and Rep78 bind specifically to the hairpin conformation of the AAV ITR and possess several enzyme activities required for resolving replication at the AAV termini. Rep52 and Rep40 have none of these properties. Reports by C. Hxc3x6lscher et al. (1994, J. Virol. 68:7169-7177; and 1995, J. Virol. 69:6880-6885) have suggested that expression of Rep78 or Rep 68 may in some circumstances be sufficient for infectious particle formation.
The Rep proteins, primarily Rep78 and Rep68, also exhibit pleiotropic regulatory activities including positive and negative regulation of AAV genes and expression from some heterologous promoters, as well as inhibitory effects on cell growth (Tratschin et al., 1986, Mol. Cell. Biol. 6:2884-2894; Labow et al., 1987, Mol. Cell. Biol., 7:1320-1325; Khleif et al., 1991, Virology, 181:738-741). The AAV p5 promoter is negatively auto-regulated by Rep78 or Rep68 (Tratschin et al., 1986, Mol. Cell. Biol. 6:2884-2894). Due to the inhibitory effects of expression of rep on cell growth, constitutive expression of rep in cell lines has not been readily achieved. For example, Mendelson et al. (1988, Virology, 166:154-165) reported very low expression of some Rep proteins in certain cell lines after stable integration of AAV genomes.
The capsid proteins VP1, VP2, and VP3 share a common overlapping sequence, but VP1 and VP2 contain additional amino terminal sequences. All three proteins are encoded by the same cap gene reading frame typically expressed from a spliced 2.3 kb mRNA transcribed from the p40 promoter. VP2 and VP3 can be generated from this mRNA by use of alternate initiation codons. Generally, transcription from p40 yields a 2.6 kb precursor mRNA which can be spliced at alternative sites to yield two different transcripts of about 2.3 kb. VP2 and VP3 can be encoded by either transcript (using either of the two initiation sites), whereas VP1 is encoded by only one of the transcripts. VP3 is the major capsid protein, typically accounting for about 90% of total virion protein. VP1 is coded from a minor mRNA using a 3xe2x80x2 donor site that is 30 nucleotides upstream from the 3xe2x80x2 donor used for the major mRNA that encodes VP2 and VP3. All three proteins are required for effective capsid production. Mutations which eliminate all three proteins (Cap-negative) prevent accumulation of single strand progeny AAV DNA, whereas mutations in the VP1 amino-terminus (xe2x80x9cLip-negativexe2x80x9d or xe2x80x9cInf-negativexe2x80x9d) can permit assembly of single-stranded DNA into particles but the infectious titer is greatly reduced.
The genetic analysis of AAV that was highlighted above was largely based upon mutational analysis of AAV genomes cloned into bacterial plasmids. In early work, molecular clones of infectious genomes of AAV were constructed by insertion of double-strand molecules of AAV into plasmids by procedures such as GC-tailing (Saimulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct, blunt-end ligation (Senapathy and Carter, 1984, J. Biol. Chem., 259:46614666). Transfection of such AAV recombinant plasmids into mammalian cells that were also infected with an appropriate helper virus, such as adenovirus, resulted in rescue and excision of the AAV genome free of any plasmid sequence, replication of the rescued genome and generation of progeny infectious AAV particles. This provided the basis for performing genetic analysis of AAV as summarized above and permitted construction of AAV transducing vectors.
Based on the genetic analysis, the general principles of AAV vector construction were defined as reviewed recently (Carter, 1992, Current Opinions in Biotechnology, 3:533-539; Muzyczka, 1992, Curr. Topics in Microbiol. and Immunol., 158:97-129). AAV vectors are generally constructed in AAV recombinant plasmids by substituting portions of the AAV coding sequence with foreign DNA to generate a recombinant AAV (rAAV) vector or xe2x80x9cpro-vectorxe2x80x9d. In the vector, the terminal (ITR) portions of the AAV sequence must generally be retained intact because these regions are generally required in cis for several functions, including excision from the plasmid after transfection, replication of the vector genome and integration and rescue from a host cell genome. In some situations, providing a single ITR may be sufficient to carry out the functions normally associated with two wild-type ITRs (see, e.g., Samulski et al., WO 94/13788, published Jun. 23, 1994).
The vector can then be packaged into an AAV particle to generate an AAV transducing virus by transfection of the vector into cells that are infected by an appropriate helper virus such as adenovirus or herpesvirus; provided that, in order to achieve replication and encapsidation of the vector genome into AAV particles, the vector must generally be complemented for any AAV functions required in trans, particularly rep and cap, that were deleted in construction of the vector.
Such AAV vectors are among a small number of recombinant virus vector systems which have been shown to have utility as in vivo gene transfer agents (reviewed in Carter, 1992, Current Opinion in Biotechnology, 3:533-539; Muzyczka, 1992, Curr. Top. Microbiol Immunol. 158:97-129) and thus are potentially of great importance for human gene therapy. AAV vectors are capable of high-frequency transduction and expression in a variety of cells including cystic fibrosis (CF) bronchial and nasal epithelial cells (see, e.g., Flotte et al., 1992a, Am. J. Respir. Cell Mol. Biol. 7:349-356; Egan et al., 1992, Nature, 358:581-584; Flotte et al., 1993a, J. Biol. Chem. 268:3781-3790; and Flotte et al., 1993b, Proc. Natl. Acad. Sci. USA, 93:10163-10167); human bone marrow-derived erythroleukemia cells (see, e.g., Walsh et al., 1992, Proc. Natl. Acad. Sci. USA, 89:7257-7261); as well as brain, eye and muscle cells. AAV may not require active cell division for transduction and expression which would be another clear advantage over retroviruses, especially in tissues such as the human airway epithelium where most cells are terminally differentiated and non-dividing.
There are at least two desirable features of any AAV vector designed for use in human gene therapy. The first is that the transducing vector be generated at titers sufficiently high to be practicable as a delivery system. This is especially important for gene therapy stratagems aimed at in vivo delivery of the vector. For example, it is likely that for many desirable applications of AAV vectors, such as treatment of cystic fibrosis by direct in vivo delivery to the airway, the desired dose of transducing vector may be from 108 to 1010, or, in some cases, in excess of 1010 particles. Secondly, the vector preparations are preferably essentially free of wild-type AAV virus (or any replication-competent AAV). The attainment of high titers of AAV vectors has been difficult for several reasons including preferential encapsidation of wild-type AAV genomes (if they are present or generated by recombination), and the difficulty in generating sufficient complementing functions such as those provided by the wild-type rep and cap genes. Useful cell lines expressing such complementing functions have been especially difficult to generate, in part because of pleiotropic inhibitory functions associated with the rep gene products. Thus, cell lines in which the rep gene is integrated and expressed may grow slowly or express rep at very low levels.
The first AAV vectors described contained foreign reporter genes such as neo, cat or dhfr expressed from AAV transcription promoters or an SV40 promoter (Tratschin et al., 1984b, Mol. Cell. Biol. 4:2072-2081; Hermonat and Muzyczka, 1984, Proc. Natl. Acad. Sci. USA, 81:6466-6470; Tratschin et al., 1985, Mol. Cell. Biol. 5:3251-3260; McLaughlin et al., 1988, J. Virol., 62:1963-1973; Lebkowski et al., 1988 Mol. Cell. Biol., 7:349-356). These vectors were packaged into AAV-transducing particles by co-transfection into adenovirus-infected cells together with a second xe2x80x9cpackaging plasmidxe2x80x9d containing the AAV rep and cap genes expressed from the wild-type AAV tanscription promoters. Several strategies have been employed in attempts to prevent encapsidation of the packaging plasmid. In some cases, (Hermonat and Muzyczka, 1984; McLaughlin et al., 1988) a large region of bacteriophage lambda DNA was inserted into the packaging plasmid within the AAV sequence to generate an oversized genome that could not be packaged. In other cases, (Tratschin et al., 1984b; Tratschin et al., 1985, Lebkowski et al., 1988), the packaging plasmid had deleted the ITR regions of AAV so that it could not be excised and replicated and thus could not be packaged. All of these approaches failed to prevent generation of particles containing replication-competent AAV DNA and also failed to generate effective high titers of AAV transducing particles. Indeed, titers of not more than 104 infectious particles per ml were cited by Hermonat and Muzyczka, 1984.
In many studies, the presence of overlapping homology between AAV sequences present in the vector and packaging plasmids resulted in the production of replication-competent AAV particles. It was shown by Senapathy and Carter (1984, J. Biol. Chem. 259:4661-4666) that the degree of recombination in such a system is approximately equivalent to the degree of sequence overlap. It was suggested in a review of the early work (Carter 1989, Handbook of Parvoviruses, Vol. II, pp. 247-284, CRC Press, Boca Raton, Fla.) that titers of 106 infectious particles per ml might be obtained, but this was based on the above-cited studies in which large amounts of replication-competent AAV contaminated the vector preparation. Such vector preparations containing replication-competent AAV will generally not be preferred for human gene therapy. Furthermore, these early vectors exhibited low transduction efficiencies and did not transduce more than 1 or 2% of cells in cultures of various human cell lines even though the vectors were supplied at multiplicities of up to 50,000 particles per cell. This may have reflected in part the contamination with replication-competent AAV particles and the presence of the AAV rep gene in the vector. Furthermore, Samulski et al. (1989, J. Virol. 63:3822-3828) showed that the presence of wild-type AAV significantly enhanced the yield of packaged vector. Thus, in packaging systems where the production of wild-type AAV is eliminated, the yield of packaged vector may actually be decreased. Nevertheless, for use in any human clinical application it will be preferable to essentially eliminate production of replication-competent AAV.
Additional studies (McLaughlin et al., 1988; Lebkowski et al., 1988) attempting to generate AAV vectors lacking the AAV rep or cap genes still generated replication-competent AAV and still produced very low transduction frequencies on human cell lines. Thus, McLaughlin et al., 1988 reported that AAV rep-negative cap-negative vectors containing the neo gene packaged with the same packaging plasmid used earlier by Hermonat and Muzyczka (1984) still contained replication-competent AAV. As a consequence, it was only possible to use this virus at a multiplicity of 0.03 particles per cell (i.e., 300 infectious units per 10,000 cell) to avoid double hits with vector and wild-type particles. Thus, when 32,000 cells were infected with 1000 infectious units, an average of 800 geneticin-resistant colonies was obtained. Although this was interpreted as demonstrating that the virus was capable of yielding a transduction frequency of 80%, in fact only 2.5% of the cells were transduced. Thus the effectively useful titer of this vector was limited. Furthermore, this study did not demonstrate that the actual titer of the vector preparation was any higher than those obtained previously by Hermonat and Muzyczka (1984). Similarly, Lebkowski et al., 1988, packaged AAV vectors which did not contain either a rep or cap gene, using an ori-negative packaging plasmid (pBa1A) identical to that used earlier by Tratschin et al., (1984b, 1985), and reported transduction frequencies that were similarly low, in that for several human cell lines not more than 1% of the cells could be transduced to geneticin resistance even with their most concentrated vector stocks. Lebkowski et al., (1988) did not report the actual vector titers in a meaningful way but the biological assays, showing not more than 1% transduction frequency when 5xc3x97106 cells were exposed to three ml of vector preparation, indicate that the titer was less than 2xc3x97104 geneticin resistant units per ml. Also, the pBa1A packaging plasmid contains overlapping homology with the ITR sequence in the vector and can lead to generation of replication-competent AAV by homologous recombination.
Laface et al. (1988) used the same vector as that used by Hermonat and Muzyczka (1984) prepared in the same way and obtained a transduction frequency of 1.5% in murine bone marrow cultures, again showing very low titer.
Samulski et al. (1987, J. Virol., 61:3096-3101) constructed a plasmid called pSub201 which contained an intact AAV genome in a bacterial plasmid but which had a deletion of 13 nucleotides at the extremity of each ITR and thus was rescued and replicated less efficiently than other AAV plasmids that contained the entire AAV genome. Samulski et al. (1989, J. Virol., 63:3822-3828) constructed AAV vectors based on pSub201 but deleted for rep and cap and containing either a hyg or neo gene expressed from an SV40 early gene promoter. They packaged these vectors by co-transfection with a packaging plasmid called pAAV/Ad which consisted of the entire AAV nucleotide sequence from nucleotide 190 to 4490 enclosed at either end with one copy of the adenovirus ITR. In this packaging plasmid the AAV rep and cap genes were expressed from their native AAV promoters (i.e. p5, p19 and p40, as discussed above). The function of the adenovirus ITR in pAAV/Ad was thought to enhance the expression level of AAV capsid proteins. However, rep is expressed from its homologous promoter and is negatively regulated and thus its expression is limited. Using their encapsidation system, Samulski et al. generated AAV vector stocks that were substantially free of replication-competent AAV but had transducing titers of only 3xc3x97104 hygromycin-resistant units per ml of supernatant. When a wild-type AAV genome was used in the packaging plasmid, the titer of the AAV vector prep was increased to 5xc3x97104 hygromycin-resistant units per ml. The low titer produced in this system thus appears to have been due in part to the defect in the ITR sequences of the basic pSub201 plasmid used for vector construction and in part due to limiting expression of AAV genes from pAAV/Ad. In an attempt to increase the titer of the AAVneo vector preparation, Samulski et al. generated vector stocks by transfecting, in bulk, thirty 10-cm dishes of 293 cells and concentrating the vector stock by banding in CsCl. This produced an AAVneo vector stock containing a total of 108 particles as measured by a DNA dot-blot hybridization assay. When this vector stock was used at multiplicities of up to 1,000 particles per cell, a transduction frequency of 70% was obtained. This suggests that the particle-to-transducing ratio is about 500 to 1,000 particles since at the ratio of one transducing unit per cell the expected proportion of cells that should be transduced is 63% according to the Poisson distribution.
Although the system of Samulski et al. (1989), using the vector plasmid pSub201 and the packaging plasmid pAAV/Ad, did not have overlapping AAV sequence homology between the two plasmids, there is overlapping homology at the XbaI sites and recombination of these sites can lead to the generation of complete replication-competent AAV. That is, although overlapping homology of AAV sequence is not present, the complete AAV sequence is contained within the two plasmids and the plasmids share a short (non-AAV) sequence that might facilitate recombination to generate replication-competent AAV, which is undesirable. That this class of recombination occurs in AAV plasmids was shown by Senapathy and Carter (1984, J. Biol. Chem. 259:466-4666). Given the problems of low titer, and the capability of generating wild-type recombinants, the system described by Samulski et al., 1989, does not have practical utility for human gene therapy.
Several other reports have described AAV vectors. For example, Srivastiva et al., (1989, Proc. Natl. Acad. Sci. USA, 86:8078-8082) described an AAV vector based on the pSub201 plasmid of Samulski et al. (1987), in which the coding sequences of AAV were replaced with the coding sequences of another parvovirus, B19. This vector was packaged into AAV particles using the pAAV/Ad packaging plasmid to generate a functional vector, but titers were not reported. This system was based on pSub201 and thus suffers from the defect described above for this plasmid. Second, the vector and the packaging plasmid contained overlapping AAV sequences (the ITR regions) and thus recombination yielding contaminating wild-type virus is highly likely.
Chatterjee et al. (1991, Vaccines 91, Cold Spring Harbor Laboratory Press, pp. 85-89), Wong et al. (1991, Vaccines 91, Cold Spring Harbor Laboratory Press, pp. 183-189), and Chatterjee et al. (1992, Science, 258:1485-1488) describe AAV vectors designed to express antisense RNA directed against infectious viruses such as HIV or Herpes simplex virus. However, these authors did not report any titers of their AAV vector stocks. Furthermore, they packaged their vectors using an ori-negative packaging plasmid analogous to that used by Tratschin et al. (1984b, 1985) containing the Ba1A fragment of the AAV genome and therefore their packaging plasmid contained AAV vector sequences that have homology with AAV sequences that were present in their vector constructs. This will also lead to generation of replication-competent AAV. Thus, Chatteijee et al., and Wong et al., used a packaging system known to give only low titer and which can lead to generation of replication-competent AAV genomes because of the overlapping homology in the vector and packaging sequences.
Other reports have described the use of AAV vectors to express genes in human lymphocytes (Muro-Cacho et al., 1992, J. Immunotherapy, 11:231-237) or a human erythroid leukemia cell line (Walsh et al., 1992, Proc. Natl. Acad. Sci. USA, 89:7257-7261) with vectors based on the pSub201 vector plasmid and pAAV/Ad packaging plasmid. Again, titers of vector stocks were not reported and were apparently low because a selective marker gene was used to identify those cells that had been successfully transduced with the vector.
Transduction of human airway epithelial cells, grown in vitro from a cystic fibrosis patient, with an AAV vector expressing the selective marker gene neo from the AAV p5 promoter was reported (Flotte et al., 1992, Am. J. Respir. Cell. Mol. Biol. 7:349-356). In this study the AAVneo vector was packaged into AAV particles using the pAAV/Ad packaging plasmid. Up to 70% of the cells in the culture could be transduced to geneticin resistance and the particle-to-transducing ratio was similar to that reported by Samulski et al. (1989). Thus to obtain transduction of 70% of the cells, a multiplicity of up to several hundred vector particles per cell was required. Transduction of human airway epithelial cells in in vitro culture using an AAV transducing vector that expressed the cystic fibrosis transmembrane conductance regulator (CFTR) gene from the AAV ITR promoter showed that the cells could be functionally corrected for the electrophysiological defect in chloride channel function that exists in cells from cystic fibrosis patients (Egan et al., Nature, 1992, 358:581-584; Flotte et al., J. Biol. Chem. 268:3781-3790).
The above-cited studies suggest that AAV vectors have potential utility as vectors for treatment of human disease by gene therapy. However, the difficulty in generating sufficient amounts of AAV vectors has been a severe limitation on the development of human gene therapy using AAV vectors. One aspect of this limitation is that there have been very few studies using AAV vectors in in vivo animal models (see, e.g., Flotte et al., 1993b; and Kaplitt et al., 1994, Nature Genetics 8:148-154). This is generally a reflection of the difficulty associated with generating sufficient amounts of AAV vector stocks having a high enough titer to be useful in analyzing in vivo delivery and gene expression.
One of the limiting factors for AAV gene therapy has been the relative inefficiency of the vector packaging systems that have been used. In the absence of suitable cell lines expressing sufficient levels of the AAV trans complementing functions, such as rep and cap, packaging of AAV vectors has been achieved in adenovirus-infected cells by co-transfection of a packaging plasmid and a vector. The efficiency of this process is expected to be limited by the efficiency of transfection of each of the plasmid constructs, and by the low level of expression of Rep proteins from the packaging plasmids described to date. Each of these problems appears to relate to the biological activities of the AAV Rep proteins which are known to be associated with pleiotropic inhibitory effects. In addition, as noted above, all of the packaging systems described above have the ability to generate replication-competent AAV by recombination.
The difficulty in generating cell lines stably expressing functional Rep apparently reflects a cytotoxic or cytostatic. function of Rep as shown by the inhibition, by Rep protein, of neo-resistant colony formation (Labow et al., 1987; Trempe et al., 1991). This also appears to relate to the tendency of Rep to reverse the immortalized phenotype in cultured cells, which has made the production of cell lines stably expressing functional rep extremely difficult. Several attempts to generate cell lines expressing rep have been made. Mendelson et al., (1988, Virology, 166:154-165) reported obtaining in one cell line some low level expression of AAV Rep52 protein but no Rep78 or Rep68 protein after stable transfection of HeLa or 293 cells with plasmids containing an AAV rep gene. Because of the absence of Rep78 and Rep68 proteins, vector could not be produced in the cell line. Another cell line made a barely detectable amount of Rep78 which was nonfunctional.
Vincent et al. (1990, Vaccines 90, Cold Spring Harbor Laboratory Press, pp. 353-359) attempted to generate cell lines containing !he AAV rep and cap genes expressed from the normal AAV promoters, but these attempts were not successful either because the vectors were contaminated with a 100-fold excess of wild-type AAV particles or because the vectors were produced at only very low titers of less than 4xc3x97103 infectious particles.
Other variations that have been proposed include systems based on the production of AAV Cap proteins that might be used to reconstitute AAV particles, e.g. by assembly in vitro (see, e.g., WO 96/00587, published Nov. 1, 1996); systems employing AAV rep-cap genes on a helper virus (see, e.g., WO 95/06743, published Mar. 9, 1995); and systems employing helper viruses from non-human mammals (see, e.g., WO 95/20671, published Aug. 3, 1995).
In yet another approach, Lebkowski et al. (U.S. Pat. No. 5,173,414, issued Dec. 22, 1992) constructed cell lines containing AAV vectors in an episomal plasmid. These cell lines could then be infected with adenovirus and transfected with the trans-complementing AAV functions rep and cap to generate preparations of AAV vector. It is claimed that this allows higher titers of AAV stocks to be produced. However, in the examples described, the only information relative to titer that is shown is that one human cell line, K562, could be transduced at efficiencies of only 1% or less, which does not indicate high titer production of any AAV vector. In this system the vector is carried as an episomal (unintegrated) construct, and it is stated that integrated copies of the vector are not preferred. In a subsequent patent (U.S. Pat. No. 5,354,678, issued Oct. 11, 1994), Lebkowski et al. suggest introducing rep and cap genes into the cell genome but the method again requires the use of episomal AAV transducing vectors comprising an Epstein-Barr virus nuclear antigen (EBNA) gene and an Epstein-Barr virus latent origin of replication; and, again, the only information relative to titer indicated that it was fairly low. Similarly, Kotin et al. (WO95/14771, published Jun. 1, 1995) suggested a system employing xe2x80x9cfirstxe2x80x9d and xe2x80x9csecondxe2x80x9d vectors to provide a source of an rAAV vector and AAV rep-cap genes, respectively. The proposed system involves a series of sequential transfections/infections of the host cells, in a transient transfection system. No data were provided regarding rAAV viral titers obtained and, indeed, it is not apparent that any rAAV virus was actually produced according to the suggested system, much less at high titer).
The problem of suboptimal levels of rep expression after plasmid transfection also relates to another biological activity of these proteins. There is evidence (Tratschin et al., 1986, Mol. Cell. Biol. 6:2884-2894) that AAV Rep proteins down-regulate their own expression from the AAV-p5 promoter which has been used in the various previously described packaging constructs such as pAAV/Ad (Samulski et al., 1989) or pBa1A (Lebkowski et al., 1988, 1992).
Another attempt to develop cell lines expressing functional rep activity was recently published by Hxc3x6lscher et al. (1994, J. Virol. 68:7169-7177). They described the generation of cell lines in which rep was placed under control of a glucocorticoid-responsive MMTV promoter. Although they observed particle formation, the particles were apparently noninfectious. Additional experiments indicated that the defect was quite fundamental; namely, there was virtually no accumulation of single-stranded rAAV DNA in the cells. Production of infectious particles required an additional transient transfection with constitutive highly-expressed rep constructs (i.e. they had to xe2x80x9cadd backxe2x80x9d Rep activity to cells that were supposed to be able to provide it themselves).
There is a significant need for methods that can be used to efficiently generate rAAV vectors that are essentially free of wild-type or other replication-competent AAV; and a corresponding need for cell lines that can be used to effectively generate such rAAV vectors. Several improved approaches to generating AAV packaging cell lines have also been described recently, see, e.g., T. Flotte et al., WO 95/13365 (Targeted Genetics Corporation and Johns Hopkins University), and corresponding U.S. Pat. No. 5,658,776; J. Trempe et al., WO 95/13392 (Medical College of Ohio), and corresponding U.S. patent application Ser. No. 08/362,608, now issued as U.S. Pat. No. 5,837,484; and J. Allen, WO 96/17947 (Targeted Genetics Corporation). The present invention provides additional improvements in the production of high-titer rAAV vector preparations.
The present invention provides compositions and methods that provide amplifiable expression of the AAV rep and/or cap genes (also referred to herein as xe2x80x9cAAV packaging genesxe2x80x9d) which can be employed in the generation of recombinant AAV (rAAV) vectors. In particular, the inventors have found that by removing the AAV rep and/or cap genes from their normal environment (i.e. flanked by the AAV ITRs) and placing them in amplifiable linkage with one or more activating elements (exemplified by the xe2x80x9cP1xe2x80x9d sequence of human chromosome 19, or analogous elements), it is possible to obtain controlled but highly amplifiable expression of the AAV packaging genes in cells to be used for the preparation of rAAV vectors. As described and exemplified herein, packaging cassettes comprising rep and/or cap sequences in amplifiable linkage to P1 or a P1-like element can be integrated into the chromosome of a host cell or can be maintained extrachromosomally as an episome. The methods and compositions of the present invention can be used to generate stable AAV producer cells that are capable of supporting production of a very large burst of rAAV particles upon infection with a suitable helper virus (such as adenovirus) or provision of helper functions.
Accordingly, in one embodiment, the invention provides a recombinant polynucleotide sequence encoding an adeno-associated virus (AAV) packaging cassette comprising at least one AAV packaging gene amplifiably linked to a P1 sequence, or an equivalent activating element.
In additional embodiments, the invention provides methods for producing high-titer stocks of rAAV vectors containing a foreign gene of interest, by co-expressing an rAAV vector containing a gene of interest along with an AAV packaging cassette comprising at least one AAV packaging gene amplifiably linked to an activating element.
The invention also provides compositions and methods for producing cell lines comprising an AAV packaging cassette of the invention together with an rAAV vector containing a gene of interest; cell lines produced thereby; compositions and methods for high-efficiency packaging of an rAAV vector containing a gene of interest; and rAAV vectors packaged according to the method of the invention.
As illustrated below, AAV packaging cassettes comprising one or more activating elements and one or more AAV packaging genes can be introduced into a host cell and propagated episomally or they can be integrated into a chromosome of a mammalian host cell. Thus, in an exemplary embodiment, the invention provides AAV packaging cassettes comprising AAV packaging genes and an activating element that are capable of integrating into the genome of a host cell (such as a mammalian cell); as well as packaging cells comprising such stably-integrated integrated cassettes. In another exemplary embodiment, the invention provides episomal packaging cassettes comprising one or more AAV packaging genes and one or more activating elements, present within a host cell as a freely-replicating episome (or capable of being introduced into a host cell such that, after introduction into the host cell, the packaging cassette will exist as a freely-replicating episomal element); as well as packaging cells comprising such episomally-maintained packaging cassettes. Illustrative examples of the design and use of both types are provided herein.